The optimal method for evaluating fluorescence microscope has not been proposed. In this study we developed two quantitative methods using biological and non-biological materials as standard specimens. Based on these methods, a new water immersion lens was tested and its high sensitivity to fluorescence was demonstrated.

First, particles of a fluorescent glass (Luminass-1; Sumita Optical Glass) were used for the standard specimen. Particles with a few micrometer diameter were embedded in silicon oil on a quartz slide glass covered with a quartz glass slip and then observed. Under the fluorescence microscope, the spectra of emission light from the excited area with or without the particle were measured by a monochromator and a photomultiplier tube (PMT). The signal-to-background ratio (S/B) was calculated from these spectra (Fig. 1, Table 1). This specimen showed a significant advantage due to less photobleaching that permits a stable and long observation time.